Combined transcriptomic and metabolomic analysis of the mechanism by which Bacillus velezensis induces resistance to anthracnose in walnut.

Walnut (Juglans spp.), a significant deciduous tree of economic and ecological importance, faces substantial threats from walnut anthracnose, primarily caused by Colletotrichum gloeosporioides. Bacillus velezensis has shown promise in mitigating this fungal pathogen. To delve deeper into the induction mechanism of B. velezensis on walnut plant resistance, we conducted a metabolomic analysis on walnut leaves from six different treatment groups. Specifically, the groups were defined as follows: Group B.v. was inoculated with B. velezensis alone, Group CK served as the blank control, and Group C.g. was inoculated solely with C. gloeosporioides. Group B.v.-C.g. received B. velezensis followed by C. gloeosporioides inoculation. Group B.v.+C.g. underwent simultaneous inoculation with both B. velezensis and C. gloeosporioides, while Group C.g.-B.v. was treated first with C. gloeosporioides then B. velezensis. A total of 1,503 metabolites were detected, mainly including flavonoids, terpenoids, and steroids. The results revealed that B. velezensis spraying not only enhanced the inherent resistance of walnut plants but also significantly regulated walnut plants already infected with C. gloeosporioides. This was mainly achieved by inducing walnut plants to adjust their metabolic pathways such as salicylic acid, jasmonic acid, and abscisic acid, thereby strengthening their stress response. Transcriptomic and metabolomic correlation analyses showed that in the comparisons of B.v. vs. CK, C.g. vs. CK, and C.g.-B.v. vs. C.g., 59, 244, and 122 differential abundance metabolites were detected, along with 7860, 3677, and 5587 differential genes, respectively. Amino acid synthesis, starch and sucrose metabolism, photosynthesis, phenylpropane metabolism, purine metabolism, and glutathione metabolism played crucial roles in walnut's disease resistance mechanism. Further analysis revealed that B. velezensis induced walnut plants to regulate multiple genes, such as LOC109005403, LOC108985444 and LOC118344177, resulting in the production of defensive metabolites such as palmitic acid, coumarin and ferulic acid, thereby enhancing their resistance to C. gloeosporioides. In summary, B. velezensis induces systemic resistance in walnut plants by modulating the metabolic pathways of salicylic acid, jasmonic acid, and abscisic acid. It enhances this resistance by strengthening cell walls, synthesizing defensive secondary metabolites, and regulating energy metabolism and stress responses. These findings provide a solid theoretical foundation for the future field application of B. velezensis in controlling walnut anthracnose.

The yeast Wickerhamomyces anomalus acts as a predator of the olive anthracnose-causing fungi, Colletotrichum nymphaeae, C. godetiae, and C. gloeosporioides.

Olive tree anthracnose is caused by infection with Colletotrichum fungi, which in Portugal are mostly C. nymphaeae, C. godetiae, and C. gloeosporioides s.s. Severe economic losses are caused by this disease that would benefit from a greener and more efficient alternative to the present agrochemical methods. Yeasts are serious candidates for pre-harvest/in field biocontrol of fungal infections. This work identified the yeast Wickerhamomyces anomalus as a strong antagonizer of the three fungi and studied in vitro this ability and its associated mechanisms. Antagonism was shown to not depend on the secretion of volatile compounds (VOCs), or siderophores or any other agar-diffusible compound, including hydrolytic enzymes. Rather, it occurred mostly in a cell-to-cell contact dependent manner. This was devised through detailed microscopic assessment of yeast-fungus cocultures. This showed that W. anomalus antagonism of the three Colletotrichum proceeded through (i) the adhesion of yeast cells to the phytopathogen hyphae, (ii) the secretion of a viscous extracellular matrix, and (iii) the emptying of the hyphae. Yeasts ultimately putatively feed on hyphal contents, which is supported by light microscopy observation of MB and PI co-culture-stained samples. Accordingly, numerous W. anomalus cells were observed packing inside C. godetiae emptied hyphae. This behaviour can be considered microbial predation and classified as necrotrophic mycoparasitism, more explicitly in the case of C. godetiae. The results support the prospect of future application of W. anomalus as a living biofungicide/BCA in the preharvest control of olive anthracnose.

First report of leaf spot on Gleditsia sinensis caused by Colletotrichum gloeosporioides in China.

Gleditsia sinensis Lam (Lamarck et al., 1788) is an endemic species widely distributed in China. In Sep. 2022, leaf spot symptoms were observed on G. sinensis in Xuhui district (31◦9'16''N, 121◦26'36''E), Shanghai, China, with an incidence rate of 55% in the examination of 9 trees. The leaves showed typical symptoms of anthracnose with irregular gray-brown spots and sunken areas. For isolation, 5 × 5 mm sections were cut from the lesion edge of 20 infected leaves collected from 2 trees. The surface of the sections was sterilized by immersion in 75% ethanol for 30 s, followed by 5% NaClO for 1 min, rinsed three times with sterile water, and dried on sterile filter paper. These sections were placed on PDA plates incubated at 25°C in darkness. Eighteen isolates with similar colony morphology were obtained and purified by single spore culturing. Two isolates (YKY2301, 2302) from separate trees were further tested. On the 6th day, the colonies had a diameter of 7.6 to 8.4 cm and appeared white to gray-white with aerial hyphae. The colony's central part exhibited an orange hue due to the conidia accumulation, while the undersides displayed an orange-yellow color. The hyphae were hyaline and smooth, with septa and branches, and the conidia were cylindrical with blunt to slightly rounded ends, measuring 13.1 to 18.8 (average 15.9) µm× 4.0 to 6.6 (average 5.4) µm (n=184). From conidia germinated on glass slides, the appressoria measured 5.5 to 6.3 µm ×4.9 to 5.1 µm (n=50) and were nearly spherical or elliptical in shape. These characteristics matched those of the Colletotrichum gloeosporioides species complex (Cannon et al., 2012; Weir et al., 2012). For molecular identification, the genomic DNA was extracted using a modified CTAB method (Luo et al., 2012). Gene fragments including ITS (PP125667, PP125668), GAPDH (PP153428, PP153429), ACT (PP153424, PP153425), TUB2(PP153917, PP190256), and ApMAT (PP153426, PP153427) were obtained by PCR using universal primers (Huang et al., 2022) and sequenced. The sequences exhibited 98.19% to 99.82% identity with the corresponding gene of the type strain C. gloeosporioides IMI356878 (JX010152, JX010056, JX009531, JX010445, JQ807843) in NCBI BLAST. A multilocus Maximum likelihood phylogenetic tree was constructed based on concatenated the five genes by PhyloSuite. It showed that YKY2301, 2302 were on the same branch with C. gloeosporioides. Based on these results, the isolates were identified as C. gloeosporioides. Pathogenicity tests were conducted by mycelial and conidia inoculation. 5 mm mycelial or blank agar plugs were inoculated onto the leaves of 2 healthy trees in a garden (25 to 30 °C), with and without wounds made by toothpick pricking (n≥3 per group). All mycelial inoculated leaves showed leaf spots on the 6th day. Three healthy 2-year-old seedlings were inoculated with either conidia (108 conidia/ml) or water by leaf spray, and maintained in a climate chamber (27 °C, 80% humidity). Inoculated seedlings showed necrotic leaf spots on day 14, and wilted within 3 weeks. The controls in all tests remained asymptomatic. The pathogen has been re-isolated and confirmed by sequencing, thus fulfilling Koch's postulates. This is the first report of leaf spots caused by C. gloeosporioides on G. sinensis in the world. As illustrated by the example of legume pod infection (Gerusa et al., 2019), it poses a potential threat to the fruits of G. sinensis, despite currently only affecting their ornamental value. This report provides basic information for future research.

Ectopic and transient expression of VvDIR4 gene in Arabidopsis and grapes enhances resistance to anthracnose via affecting hormone signaling pathways and lignin production.

BACKGROUND: DIR (Dirigent) proteins play important roles in the biosynthesis of lignin and lignans and are involved in various processes such as plant growth, development, and stress responses. However, there is less information about VvDIR proteins in grapevine (Vitis vinifera L). RESULTS: In this study, we used bioinformatics methods to identify members of the DIR gene family in grapevine and identified 18 VvDIR genes in grapevine. These genes were classified into 5 subfamilies based on phylogenetic analysis. In promoter analysis, various plant hormones, stress, and light-responsive cis-elements were detected. Expression profiling of all genes following Colletotrichum gloeosporioides infection and phytohormones (salicylic acid (SA) and jasmonic acid (JA)) application suggested significant upregulation of 17 and 6 VvDIR genes, respectively. Further, we overexpressed the VvDIR4 gene in Arabidopsis thaliana and grapes for functional analysis. Ectopic expression of VvDIR4 in A. thaliana and transient expression in grapes increased resistance against C. gloeosporioides and C. higginsianum, respectively. Phenotypic observations showed small disease lesions in transgenic plants. Further, the expression patterns of genes having presumed roles in SA and JA signaling pathways were also influenced. Lignin contents were measured before and after C. higginsianum infection; the transgenic A. thaliana lines showed higher lignin content than wild-type, and a significant increase was observed after C. higginsianum infection. CONCLUSIONS: Based on the findings, we surmise that VvDIR4 is involved in hormonal and lignin synthesis pathways which regulate resistance against anthracnose. Our study provides novel insights into the function of VvDIR genes and new candidate genes for grapevine disease resistance breeding programs.

Resistant risk and resistance mechanism of florylpicoxamid in Colletotrichum gloeosporioides isolated from Chinese walnut.

Colletotrichum gloeosporioides is the causal pathogen for the devastating walnuts anthracnose. A novel quinone inside inhibitor (QiI) fungicide florylpicoxamid has strong inhibitory efficacy against C. gloeosporioides. This study looked into the resistance risk and mechanism of C. gloeosporioides to florylpicoxamid. The basal level sensitivity of C. gloeosporioides isolates (n = 102) to florylpicoxamid was established with an average 50% mycelial growth inhibition concentration (EC50) value of 0.069 ± 0.035 µg/mL. Six stable florylpicoxamid-resistant mutants with resistance factors of >1000 were produced. The fitness of every mutant was much lower than that of their parental isolates. In general, the resistance risk of C. gloeosporioides to florylpicoxamid would be moderate. Molecular docking results revealed that the amino acid substitutions A37V, and S207L in CgCytb lead to a reduction in the binding affinity between florylpicoxamid and CgCytb, indicating that these two mutations (S207L and A37V in CgCytb) indeed confer florylpicoxamid resistance in C. gloeosporioides. These findings offer a fresh viewpoint on the mechanism underlying QiI fungicide resistance and could support the prudent application of florylpicoxamid in the future to combat walnut anthracnose.

Inhibitory mechanism of 4-ethyl-1,2-dimethoxybenzene produced by Streptomyces albidoflavus strain ML27 against Colletotrichum gloeosporioides.

Actinomycetes have emerged as significant biocontrol resources due to their rich array of bioactive natural products. While much research has historically focused on secondary metabolites isolated from their fermentation broth, there remains a dearth of reports on their volatile organic compounds (VOCs). Here, strain ML27, isolated from soil, was identified as Streptomyces albidoflavus based on morphological features, physiological, biochemical, and molecular characteristics (16S rRNA, atpD, recA, and rpoB gene sequences). VOCs from S. albidoflavus strain ML27 were effectively captured using solid-phase microextraction (SPME) and tentatively identified through gas chromatography-mass spectrometry (GC/MS). Among these compounds, 4-ethyl-1,2-dimethoxybenzene exhibited broad-spectrum antifungal activity and demonstrated efficacy in controlling citrus anthracnose, with a control efficacy of 86.67%. Furthermore, the inhibitory mechanism of 4-ethyl-1,2-dimethoxybenzene against Colletotrichum gloeosporioides was revealed. Results indicated that 4-ethyl-1,2-dimethoxybenzene induced swelling, deformity, and breakage in C. gloeosporioides mycelia, and significantly inhibited spore germination. Transcriptome analysis revealed that 4-ethyl-1,2-dimethoxybenzene inhibited the growth and development of C. gloeosporioides primarily by disrupting energy metabolism and the integrity of the cell wall and membrane. Based on these results, it is promising to develop 4-ethyl-1,2-dimethoxybenzene as a novel biopesticide for controlling citrus anthracnose.

Antifungal Mechanism of Ruta graveolens Essential Oil: A Colombian Traditional Alternative against Anthracnose Caused by Colletotrichum gloeosporioides.

Here, we report for the first time on the mechanisms of action of the essential oil of Ruta graveolens (REO) against the plant pathogen Colletotrichum gloeosporioides. In particular, the presence of REO drastically affected the morphology of hyphae by inducing changes in the cytoplasmic membrane, such as depolarization and changes in the fatty acid profile where straight-chain fatty acids (SCFAs) increased by up to 92.1%. In addition, REO induced changes in fungal metabolism and triggered apoptosis-like responses to cell death, such as DNA fragmentation and the accumulation of reactive oxygen species (ROS). The production of essential enzymes involved in fungal metabolism, such as acid phosphatase, ß-galactosidase, ß-glucosidase, and N-acetyl-ß-glucosaminidase, was significantly reduced in the presence of REO. In addition, C. gloeosporioides activated naphthol-As-BI phosphohydrolase as a mechanism of response to REO stress. The data obtained here have shown that the essential oil of Ruta graveolens has a strong antifungal effect on C. gloeosporioides. Therefore, it has the potential to be used as a surface disinfectant and as a viable replacement for fungicides commonly used to treat anthracnose in the postharvest testing phase.

Research on the Isolation of Endophytic Fungi from Papaya and the Prevention of Colletotrichum gloeosporioides.

Endophytic fungi can be used as a source of herbal antioxidants to overcome the limitations of low yield and lengthy growth cycles associated with using plants as raw materials for antioxidant production. Papaya fruit is often susceptible to infection by Colletotrichum gloeosporioides after harvest, leading to postharvest rot. Endophytic fungi were extracted with ethyl acetate, and the initial screening concentration was 100 mg/L. Seven strains were identified, with scavenging rates exceeding 50% and strong antioxidant activity. The IC50 values in DPPH and ABTS free radical scavenging assays ranged from 19.72 to 84.06 mg/L and from 14.34 to 64.63 mg/L, respectively. Strain Y17 exhibited robust antioxidant activity (IC50 < 20 mg/L) and was identified as Penicillium rolfsii (MT729953) through ITS sequencing. Treatment of papaya fruit wounds with a fermentation broth of strain Y17 significantly inhibited the infection and colonization of anthracnose pathogens, resulting in a slowed disease incidence rate. This promoted the activity of protective enzymes, such as CAT, POD, and SOD, in the papaya fruit and slowed down the rate of MDA accumulation. This strain, which was found to have antioxidant activity in this study, has the potential to control anthracnose in papaya and has value in terms of further development and utilization.

Functional Characterization of the Transcription Factor Gene CgHox7 in Colletotrichum gloeosporioides, Which Is Responsible for Poplar Anthracnose.

Colletotrichum gloeosporioides is the main pathogen that causes poplar anthracnose. This hemibiotrophic fungus, which can severely decrease the economic benefits and ecological functions of poplar trees, infects the host by forming an appressorium. Hox7 is an important regulatory factor that functions downstream of the Pmk1 MAPK signaling pathway. In this study, we investigated the effect of deleting CgHox7 on C. gloeosporioides. The conidia of the ΔCgHox7 deletion mutant germinated on a GelBond membrane to form non-melanized hyphal structures, but were unable to form appressoria. The deletion of CgHox7 weakened the ability of hyphae to penetrate a cellophane membrane and resulted in decreased virulence on poplar leaves. Furthermore, deleting CgHox7 affected the oxidative stress response. In the initial stage of appressorium formation, the accumulation of reactive oxygen species differed between the ΔCgHox7 deletion mutant and the wild-type control. Moreover, CgHox7 expression was necessary for maintaining cell wall integrity. Considered together, these results indicate that CgHox7 is a transcription factor with crucial regulatory effects on appressorium formation and the pathogenicity of C. gloeosporioides.

A dual RNA-seq analyses revealed dynamic arms race during the invasion of walnut by Colletotrichum gloeosporioides.

BACKGROUND: Walnut anthracnose caused by Colletotrichum gloeosporioides seriously endangers the yield and quality of walnut, and has now become a catastrophic disease in the walnut industry. Therefore, understanding both pathogen invasion mechanisms and host response processes is crucial to defense against C. gloeosporioides infection. RESULTS: Here, we investigated the mechanisms of interaction between walnut fruits (anthracnose-resistant F26 fruit bracts and anthracnose-susceptible F423 fruit bracts) and C. gloeosporioides at three infection time points (24hpi, 48hpi, and 72hpi) using a high-resolution time series dual transcriptomic analysis, characterizing the arms race between walnut and C. gloeosporioides. A total of 20,780 and 6670 differentially expressed genes (DEGs) were identified in walnut and C. gloeosporioides against 24hpi, respectively. Generous DEGs in walnut exhibited opposite expression patterns between F26 and F423, which indicated that different resistant materials exhibited different transcriptional responses to C. gloeosporioides during the infection process. KEGG functional enrichment analysis indicated that F26 displayed a broader response to C. gloeosporioides than F423. Meanwhile, the functional analysis of the C. gloeosporioides transcriptome was conducted and found that PHI, SignalP, CAZy, TCDB genes, the Fungal Zn (2)-Cys (6) binuclear cluster domain (PF00172.19) and the Cytochrome P450 (PF00067.23) were largely prominent in F26 fruit. These results suggested that C. gloeosporioides secreted some type of effector proteins in walnut fruit and appeared a different behavior based on the developmental stage of the walnut. CONCLUSIONS: Our present results shed light on the arms race process by which C. gloeosporioides attacked host and walnut against pathogen infection, laying the foundation for the green prevention of walnut anthracnose.

First Report of Colletotrichum gloeosporioides Causing Leaf Blight on Cercis chinensis Bunge in China.

Cercis chinensis Bunge, commonly used as an ornamental plant, is native to southeastern China and extensively cultivated in gardens across major cities in the country. In August 2023, a new high-incidence disease was discovered at Huangshan University in Huangshan, Anhui Province, China. The symptoms initially began as small brown spots, which gradually expanded into large irregular brown spots with black-brown edges. The disease was investigated at both Jilingshan Park and Huangshan University, where C. chinensis Bunge was planted, revealing an average incidence rate of was 85 % at these sites. Seventy two leaf tissue samples (3 to 4 mm²) were collected from the margins of the lesion and subjected to surface sterilization with 75% ethanol for 30 seconds followed by 1% sodium hypochlorite for 90 seconds. Subsequently, the tissues were rinsed with sterile H2O, placed on potato dextrose agar (PDA) medium, and incubated at 25℃ for 5 days. The same fungus was isolated from 90% of the tissues, and pure cultures were obtained by monosporic isolation. Representative isolates ZJ 2-1, ZJ 2-2 and ZJ 2-3 were selected for morphological and molecular characterization. The colonies displayed a color range from white to gray, with white margins and aerial hyphae, while the reverse side of the colonies appeared gray to brown. Conidia were cylindrical, aseptate, with obtuse to slightly rounded ends, measuring 15.8±1.8×4.7±0.56 µm (n = 50). The morphological characteristics were generally consistent with those of Colletotrichum gloeosporioides species complex (Weir et al. 2012). Five conserved regions of isolates (ZJ 2-1, ZJ 2-2 and ZJ 2-3), including the internal transcribed spacer (ITS), glutamine synthase (GS), calmodulin (CAL), actin (ACT), and chitin synthase 1(CHS1) gene regions, were amplified using specific primers ITS1/ITS4 (Gardes et al. 1993), GSR1/GSF1 (Guerber et al. 2003), CL1C/CL2C (Li et al. 2018), ACT-512F/ACT-783R, and CHS-79F/CHS-345R (Zhu et al. 2019), respectively. Using the BLAST, ITS, GS, CAL, ACT and CHS1 gene sequences (GenBank accession nos. PP514751, PP448025, PP448026, PP448027 and PP448028, respectively) were 100% (594 out of 594 bp), 100% (864 out of 864 bp), 100% (299 out of 299 bp), 100% (732 out of 732 bp) and 100% (282 out of 282 bp) identical to C. gloeosporioides (GenBank accession nos. JX010152, JX010085, JX009818, JX009731 and JX009531, respectively). A Maximum Likelihood phylogenetic tree, constructed by combining all sequenced loci in MEGA7, showed that the isolates ZJ 2-1, ZJ 2-2 and ZJ 2-3 clustered within the C. gloeosporioides clade with 99% bootstrap support (Fig. S1). To fulfill Koch's postulates, five C. chinensis Bunge plants were tested for pathogenicity in the field with isolates ZJ 2-1, ZJ 2-2 and ZJ 2-3 at Huangshan University. Twelve leaves from each tree were wounded and inoculated with mycelial plugs (approximately 4 mm in diameter) and 10 µl of a spore suspension (1.0 × 106 conidia/ml) of C. gloeosporioides. Inoculation with sterile PDA plugs and pure water on leaves of each tree served as negative controls. Plastic bags were used to wrap the leaves, and sterile H2O was sprayed into the bags to maintain moisture conditions (Zhang et al.2020). The experiment was repeated two times, and within 5 days, all inoculated points displayed lesions similar to those observed in the field, whereas controls remained asymptomatic (Fig. S2). The same fungus was reisolated from these lesions with a frequency of 100%. Consequently, the pathogen responsible the disease in C. chinensis Bunge was identified as C. gloeosporioides. To the best of our knowledge, this is the first report of C. gloeosporioides causing leaf blight on C. chinensis Bunge in China. This study provides valuable insights for implementing targeted measures to control leaf blight on C. chinensis Bunge and lays a foundation for the prevention and treatment of the disease.

Fungicide resistance in Colletotrichum fructicola and Colletotrichum siamense causing peach anthracnose in China.

Peach is one of the popular and economically important fruit crops in China. Peach cultivation is hampered due to attacks of anthracnose disease, causing significant economic losses. Colletotrichum fructicola and Colletotrichum siamense belong to the Colletotrichum gloeosporioides species complex and are considered major pathogens of peach anthracnose. Application of different groups of fungicides is a routine approach for controlling this disease. However, fungicide resistance is a significant drawback in managing peach anthracnose nowadays. In this study, 39 isolates of C. fructicola and 41 isolates of C. siamense were collected from different locations in various provinces in China. The sensitivity of C. fructicola and C. siamense to some commonly used fungicides, i.e., carbendazim, iprodione, fluopyram, and propiconazole, was determined. All the isolates of C. fructicola collected from Guangdong province showed high resistance to carbendazim, whereas isolates collected from Guizhou province were sensitive. In C. siamense, isolates collected from Hebei province showed moderate resistance, while those from Shandong province were sensitive to carbendazim. On the other hand, all the isolates of C. fructicola and C. siamense showed high resistance to the dicarboximide (DCF) fungicide iprodione and succinate dehydrogenase inhibitor (SDHI) fungicide fluopyram. However, they are all sensitive to the demethylation inhibitor (DMI) fungicide propiconazole. Positive cross-resistance was observed between carbendazim and benomyl as they are members of the same methyl benzimidazole carbamate (MBC) group. While no correlation of sensitivity was observed between different groups of fungicides. No significant differences were found in each fitness parameter between carbendazim-resistant and sensitive isolates in both species. Molecular characterization of the ß-tubulin 2 (TUB2) gene revealed that in C. fructicola, the E198A point mutation was the determinant for the high resistance to carbendazim, while the F200Y point mutation was linked with the moderate resistance to carbendazim in C. siamense. Based on the results of this study, DMI fungicides, e.g., propiconazole or prochloraz could be used to control peach anthracnose, especially at locations where the pathogens have already developed the resistance to carbendazim and other fungicides.

Essential oils inhibiting Alternaria alternata and Colletotrichum gloeosporioides: a review.

Essential oils (EOs) have been investigated for their effectiveness against fungal fruit pathogens. The present review article summarises the EOs that inhibit Alternaria alternata and Colletotrichum gloeosporioides in the pre- and post-harvest stages of fruits. Thirty-nine scientific papers focusing on the extraction conditions and the antifungal activity of EOs were selected. The retrieved studies came mainly from China and Brazil. Hydrodistillation has been identified as the most used extractive method. The yields and chemical profiles were variable among the species. The in vitro studies were larger than the in vivo studies. The application of EOs reduced the incidence of fungal diseases in tomatoes (Lycopersicon esculentum), papaya (Carica papaya) and mango (Mangifera indica). EOs resulted as a potential ecological alternative for treating fungal diseases in fruits requiring further investigation.

The miR7125-MdARF1 module enhances the resistance of apple to Colletotrichum gloeosporioides by promoting lignin synthesis in response to salicylic acid signalling.

Apple is an important cash crop in China, and it is susceptible to fungal infections that have deleterious effects on its yield. Apple bitter rot caused by Colletorichum gloeosporioides is one of the most severe fungal diseases of apple. Salicylic acid (SA) is a key signalling molecule in the plant disease resistance signalling pathways. Lignin synthesis also plays a key role in conferring disease resistance. However, few studies have clarified the relationship between the SA disease resistance signalling pathway and the lignin disease resistance pathway in apple. MdMYB46 has previously been shown to promote lignin accumulation in apple and enhance salt and osmotic stress tolerance. Here, we investigated the relationship between MdMYB46 and biological stress; we found that MdMYB46 overexpression enhances the resistance of apple to C. gloeosporioides. We also identified MdARF1, a transcription factor upstream of MdMYB46, via yeast library screening and determined that MdARF1 was regulated by miR7125 through psRNATarget prediction. This regulatory relationship was confirmed through LUC and qRT-PCR experiments, demonstrating that miR7125 negatively regulates MdARF1. Analysis of the miR7125 promoter revealed that miR7125 responds to SA signals. The accumulation of SA level will result in the decrease of miR7125 expression level. In sum, the results of our study provide novel insights into the molecular mechanisms underlying the resistance of apple to C. gloeosporioides and reveal a new pathway that enhances lignin accumulation in apple in response to SA signals. These findings provide valuable information for future studies aimed at breeding apple for disease resistance.

The new CFEM protein CgCsa required for Fe3+ homeostasis regulates the growth, development, and pathogenicity of Colletotrichum gloeosporioides.

Secreted common fungal extracellular membrane (CFEM) domain proteins have been implicated in multiple biological functions in fungi. However, it is still largely unknown whether the ferric iron (Fe3+), as an important trace element, was involved with the biological function of CFEM proteins. In this study, a new CFEM protein CgCsa, with high expression levels at the early inoculation stage on peppers by Colletotrichum gloeosporioides was investigated. Deletion of the targeted gene CgCsa revealed multiple biological roles in hyphal growth restriction, highly reduced conidial yield, delayed conidial germination, abnormal appressorium with elongated bud tubes, and significantly reduced virulence of C. gloeosporioides. Moreover, in CgCsa mutants, the expression levels of four cell wall synthesis-related genes were downregulated, and cell membrane permeability and electrical conductivity were increased. Compared to the wild-type, the CgCsa mutants downregulated expressions of iron transport-related genes, in addition, its three-dimensional structure was capable binding with iron. Increase in the Fe3+ concentration in the culture medium partially recovered the functions of ΔCgCsa mutant. This is probably the first report to show the association between CgCsa and iron homeostasis in C. gloeosporioides. The results suggest an alternative pathway for controlling plant fungal diseases by deplete their trace elements.

Design and Synthesis of Eugenol Derivatives Bearing a 1,2,3-Triazole Moiety for Papaya Protection against Colletotrichum gloeosporioides.

A series of 19 novel eugenol derivatives containing a 1,2,3-triazole moiety was synthesized via a two-step process, with the key step being a copper(I)-catalyzed azide-alkyne cycloaddition reaction. The compounds were assessed for their antifungal activities against Colletotrichum gloeosporioides, the causative agent of papaya anthracnose. Triazoles 2k, 2m, 2l, and 2n, at 100 ppm, were the most effective, reducing mycelial growth by 88.3, 85.5, 82.4, and 81.4%, respectively. Molecular docking calculations allowed us to elucidate the binding mode of these derivatives in the catalytic pocket of C. gloeosporioides CYP51. The best-docked compounds bind closely to the heme cofactor and within the channel access of the lanosterol (LAN) substrate, with crucial interactions involving residues Tyr102, Ile355, Met485, and Phe486. From such studies, the antifungal activity is likely attributed to the prevention of substrate LAN entry by the 1,2,3-triazole derivatives. The triazoles derived from natural eugenol represent a novel lead in the search for environmentally safe agents for controlling C. gloeosporioides.

Combined control of plant diseases by Bacillus subtilis SL44 and Enterobacter hormaechei Wu15.

Co-incubation of plant growth promoting rhizobacteria (PGPRs) have been proposed as a potential alternative to pesticides for controlling fungal pathogens in crops, but their synergism mechanisms are not yet fully understood. In this study, combined use of Bacillus subtilis SL44 and Enterobacter hormaechei Wu15 could decrease the density of Colletotrichum gloeosporioides and Rhizoctonia solani and enhance the growth of beneficial bacteria on the mycelial surface, thereby mitigating disease severity. Meanwhile, PGPR application led to a reorganization of the rhizosphere microbial community through modulating its metabolites, such as extracellular polymeric substances and chitinase. These metabolites demonstrated positive effects on attracting and enhancing conventional periphery bacteria, inhibiting fungal pathogens and promoting soil health effectively. The improvement in the microbial community structure altered the trophic mode of soil fungal communities, effectively decreasing the proportion of saprotrophic soil and reducing fungal plant diseases. Certain combinations of PGPR have the potential to serve as precise instruments for managing plant pathogens.

Production and Physicochemical Characterization of the Gel Obtained in the Fermentation Process of Blue Corn Flour (Zea mays L.) with Colletotrichum gloeosporioides.

Food gels are viscoelastic substances used in various gelled products manufactured around the world. Polysaccharides are the most common food gelling agents. The aim of this work was the production and characterization of a gel produced in a blue corn flour fermentation process, where different proportions were used of blue corn (Zea mays L.) flour and Czapek Dox culture medium (90 mL of culture medium with 10 g of blue corn flour, 80 mL of culture medium with 20 g of blue corn flour, and 70 mL of culture medium with 30 g of blue corn flour) and were fermented for three different durations (20, 25, and 30 days) with the Colletotrichum gloeosporioides fungus. A characterization of the gel was carried out studying the rheological properties, proximal analysis, toxicological analysis, microscopic structure, and molecular characterization, in addition to a solubility test with three different organic solvents (ethanol, hexane, and ethyl acetate, in addition to water). The results obtained showed in the rheological analysis that the gel could have resistance to high temperatures and a reversible behavior. The gel is soluble in polar solvents (ethanol and water). The main chemical components of the gel are carbohydrates, especially polysaccharides, and it was confirmed by FT-IR spectroscopy that the gel may be composed of pectin.

A Lateral Flow-Recombinase Polymerase Amplification Method for Colletotrichum gloeosporioides Detection.

The greater yam (Dioscorea alata), a widely cultivated and nutritious food crop, suffers from widespread yield reduction due to anthracnose caused by Colletotrichum gloeosporioides. Latent infection often occurs before anthracnose phenotypes can be detected, making early prevention difficult and causing significant harm to agricultural production. Through comparative genomic analysis of 60 genomes of 38 species from the Colletotrichum genus, this study identified 17 orthologous gene groups (orthogroups) that were shared by all investigated C. gloeosporioides strains but absent from all other Colletotrichum species. Four of the 17 C. gloeosporioides-specific orthogroups were used as molecular markers for PCR primer designation and C. gloeosporioides detection. All of them can specifically detect C. gloeosporioides out of microbes within and beyond the Colletotrichum genus with different sensitivities. To establish a rapid, portable, and operable anthracnose diagnostic method suitable for field use, specific recombinase polymerase amplification (RPA) primer probe combinations were designed, and a lateral flow (LF)-RPA detection kit for C. gloeosporioides was developed, with the sensitivity reaching the picogram (pg) level. In conclusion, this study identified C. gloeosporioides-specific molecular markers and developed an efficient method for C. gloeosporioides detection, which can be applied to the prevention and control of yam anthracnose as well as anthracnose caused by C. gloeosporioides in other crops. The strategy adopted by this study also serves as a reference for the identification of molecular markers and diagnosis of other plant pathogens.

First Report of Anthracnose on Wurfbainia villosa var. villosa Caused by Colletotrichum gloeosporioides in China.

Wurfbainia villosa var. villosa is a traditional Chinese herbal medicine under the family Zingiberaceae, and its ripe fruits (called Fructus Amomi) are widely used clinically for the treatment of gastrointestinal disorders (Yang et al. 2023; Chen et al. 2023). In September 2023, plants of W. villosa var. villosa exhibited anthracnose-like symptoms on leaf with a disease incidence of 35% (n = 100 investigated plants) in an approximately 90 m2 field in Guangning, China (N23°42'51.70″, E112°26'35.75″). Light yellowish-green spots (~2 mm diameter) initially appeared on the infected leaves, gradually formed sub-circular or irregular spots, then fused and expanded, resulting in wilting of the leaves. To identify the causal agent, 10 symptomatic leaves were collected and transferred to the laboratory. The symptomatic leaf samples were surface sterilized in 0.5% NaClO for 2 min, and in 70% ethanol for 30 s, then washed three times with sterile water and air-dried on sterile filter paper. The leaf tissues were placed on potato dextrose agar (PDA) medium containing 100 µg mL-1 of ampicillin (Sigma-Aldrich, St. Louis, MO) and incubated for 7 days at 28°C in darkness. Nine isolates with similar colony morphology were isolated from the 10 plated leaves. Three representative isolates (GNAF03, GNAF06, GNAF09 with approximately 3.5 cm in diameter after 3 days of incubation) appeared gray to dark brown with dense aerial hyphae at the front and gray to black colonies on the reverse of the plates. Conidia were cylindrical and measured 21.2 to 29.3 µm long × 7.1 to 9.6 µm wide (n = 50). Appressoria were formed by the tips of germ tubes or hyphae and were brown, ellipsoid, thick-walled, and smooth-margined, measuring 10.2 to 12.3 µm long × 6.4 to 8.2 µm wide (n = 50). Morphologically, the fungal isolates resembled Colletotrichum sp. (Weir et al. 2012). For molecular analysis, genomic DNA was extracted from fresh mycelia of the three isolates, and the primers ACT-512F/ACT-783R, CL1/CL2A, GDF/GDR, and ITS1/ITS4 were used to amplify partial regions of rDNA-ITS, actin (ACT), calmodulin (CAL), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) regions, respectively (Weir et al. 2012). The resulting sequences with more than 99% nucleotide identity to C. gloeosporioides were submitted to GenBank (accession numbers PP552725, PP552726, and OR827444 for ACT; PP552727, PP552728, and OR827443 for CAL; PP552729, PP552730, and OR827445 for GAPDH; PP549996, PP549999, and OR841394 for ITS). A phylogenetic tree was generated by the maximum likelihood method using the concatenated sequences of ACT, CAL, GADPH, and ITS by Polysuite software (Damm et al. 2020). Based on morphological and molecular analysis, the three isolates were characterized as C. gloeosporioides. The pathogenicity of the GNAF09 isolate was assessed on W. villosa var. villosa seedling leaves inoculated by spraying with 40 µL of conidial suspension at 106 conidia mL-1 or wounded with a sterile toothpick then inoculated with mycelial agar plugs (5 mm diameter). Control leaves were inoculated with 40 µL of sterile distilled water or agar plugs without mycelia. The inoculated plants were placed in a humid chamber at 28°C with 80% humidity and a 12 h light-dark photoperiod. Symptoms similar to those seen on naturally infected leaves were observed on all inoculated leaves after 7 days inoculation. Re-isolation was performed from 80% of the inoculated leaves and isolates were confirmed as C. gloeosporioides morphologically, confirming Koch's postulates, and by sequencing the ACT, CAL, GADPH, and ITS regions. The control groups remained asymptomatic. In previous studies, C. gloeosporioides has also caused anthracnose on Chinese medicinal plants, including Baishao (Radix paeoniae alba) (Zhang et al. 2017) and Rubia cordifolia L. (Tang et al. 2020). To our knowledge, this is the first report of C. gloeosporioides causing anthracnose on W. villosa var. villosa in China. The results of our report serve as valuable references for further research on this disease.